[Modulation of the intestinal microbiota by nutritional interventions]. / Modulation der intestinalen Mikrobiota durch Ernährungsinterventionen.

Humans live in symbiosis with billions of commensal bacteria. The so-called microbiota live on different biological interfaces such as the skin, the urogenital tract and the gastrointestinal tract. Commensal bacteria replace potentially pathogenic microbes, synthesize vitamins and ferment dietary fibre. An imbalance in the bacterial composition of the intestinal microbiota has been associated with various diseases including gut-associated disorders such as inflammatory bowel diseases, colorectal cancer and nonalcoholic fatty liver disease. Furthermore, a shift in the microbiota composition appears to be of pathophysiological relevance which renders the specific modulation of the intestinal microbiota a promising approach in the treatment of the above mentioned diseases. Our intestinal microbiota composition is mainly modulated by dietary macro- and micronutrients but also by secondary plant compounds and synthetic food additives such as emulsifiers and artificial sweeteners. Nutritional interventions with the purpose to modulate the intestinal microbiota show only limited therapeutic potential in the treatment of gut-associated disorders, which may be due to individual differences in the intestinal microbiota composition and a lack of specificity. A combination of newly established technical analytic approaches involving a machine-learning algorithm may bridge the currently existing limitations by providing a personalized, highly-specific and consequently therapeutically effective microbiota modulation.

Effects of a six-week intraduodenal supplementation with quercetin on liver lipid metabolism and oxidative stress in peripartal dairy cows.

The purpose of this study was to evaluate possible effects of quercetin (Q) on liver lipid metabolism and antioxidative status in periparturient dairy cows. The periparturient period is associated with enormous metabolic changes for dairy cows. Energy needs for incipient lactation are too high to be balanced by feed intake, leading to negative energy balance and body fat mobilization. It has been estimated that this leads to the development of fatty liver in about 50% of cows, which are at high risk for disease. Furthermore, the antioxidative status of these cows may be impaired. Quercetin is a plant flavonoid having hepatoprotective and antioxidative potential and the ability to reduce liver lipid accumulation in monogastric animals. Little information is available in regard to these effects in ruminants. To prevent microbial Q degradation in the rumen, Q was administered via a duodenal fistula to improve systemic availability. Five cows of the Q-treated group received, daily, 100 mg of quercetin dehydrate/kg BW in a 0.9% sodium chloride solution from d -20 until d 20 relative to calving, whereas 5 control (CTR) cows received only a sodium chloride solution. Blood samples were taken weekly and liver biopsies were performed in wk -4, -2, and 3 relative to calving. Cows treated with Q showed a tendency ( = 0.082) for lower liver fat content compared with CTR cows. Liver glycogen, glutathione concentrations, and relative mRNA abundance of genes related to hepatic lipid metabolism and antioxidative status as well as parameters of antioxidative status in plasma were not affected ( > 0.1) by Q supplementation. In conclusion, liver fat content in dairy cows tended to be reduced by Q supplementation, but potential underlying mechanisms remain unclear because analyzed parameters related to hepatic lipid metabolism and antioxidative defense were not altered by Q supplementation.

A combination of lipoic acid plus coenzyme Q10 induces PGC1α, a master switch of energy metabolism, improves stress response, and increases cellular glutathione levels in cultured C2C12 skeletal muscle cells.

Skeletal muscle function largely depend on intact energy metabolism, stress response, and antioxidant defense mechanisms. In this study, we tested the effect of a combined supplementation of α-lipoic acid (LA) plus coenzyme Q10 (Q10) on PPARγ-coactivator α (PGC1α) activity, expression of glutathione-related phase II enzymes and glutathione (GSH) levels in cultured C2C12 myotubes. Supplementation of myotubes with 250 µmol/L LA plus 100 µmol/L Q10 significantly increased nuclear levels of PGC1α, a master switch of energy metabolism and mitochondrial biogenesis. The increase of nuclear PGC1α was accompanied by an increase in PPARγ transactivation, a downstream target of PGC1α, and an increase in mitochondrial transcription factor A mRNA centrally involved in mitochondrial replication and transcription. Furthermore, supplementation of myotubes with LA plus Q10 resulted in an increase of genes encoding proteins involved in stress response, GSH synthesis, and its recycling. In LA-plus-Q10-treated myotubes a significant 4-fold increase in GSH was evident. This increase in GSH was accompanied by increased nuclear Nrf2 protein levels, partly regulating γGCS and GST gene expression. Present data suggest that the combined supplementation of skeletal muscle cells with LA plus Q10 may improve energy homeostasis, stress response, and antioxidant defense mechanisms.

Cyanidin does not affect sulforaphane-mediated Nrf2 induction in cultured human keratinocytes.

There is increasing interest in the gene-regulatory activities of isothiocyanates and flavonoids in human skin. Nrf2 agonists, such as isothiocyanate sulforaphane (SFN), have been shown to promote chemopreventive effects in skin both in vitro and in vivo. Recent data indicate that different secondary plant compounds may either antagonise or enhance SFN-induced Nrf2 activation. We therefore studied the interactions of a flavonoid, cyanidin and the potent Nrf2 inductor SFN in cultured human keratinocytes (HaCaT cells). We observed that cyanidin does not induce the activation of Nrf2 and its target genes, γ-glutamylcysteine synthetase (γGCS), NAD(P)H:quinone oxidoreductase 1 and haem oxygenase-1 in HaCaT cells. Furthermore, SFN-mediated Nrf2 activation and its target gene expression were not further enhanced by the co-application of SFN with cyanidin.

3,3'-Diindolylmethane but not indole-3-carbinol activates Nrf2 and induces Nrf2 target gene expression in cultured murine fibroblasts.

There is increasing interest in the gene-regulatory activity of Brassica vegetable derived phytochemicals such as 3,3'-diindolylmethane (DIM) and indole-3-carbinol (I3C). DIM is formed under acidic conditions by dimerization of I3C. This study compared the Nrf2 activating potential of DIM and I3C in murine fibroblasts (NIH3T3). In contrast to its precursor I3C, DIM induces the transactivation of Nrf2. Furthermore, Nrf2 targets such as HO-1, γGCS and NQO1 were increased on the mRNA and protein levels following DIM treatment. DIM was less potent than sulforaphane (used as positive control) in inducing Nrf2-dependent gene expression. The present data suggest that the dimerization of I3C to DIM increases its Nrf2 inducing activity.

Cellular uptake, stability, visualization by 'Naturstoff reagent A', and multidrug resistance protein 1 gene-regulatory activity of cyanidin in human keratinocytes.

There is increasing interest in the role of anthocyanidins as potential skin protective phytochemicals. However, little is known if and to what extent anthocyanidins are taken up by the human skin. In the present study cellular uptake (as determined by HPLC), stability, and gene-regulatory activity of cyanidin were determined in human HaCaT keratinocytes in culture. Using the fluorescent dye Naturstoff reagent A cyanidin was visualized in order to determine its cellular accumulation via flow cytometry and fluorescence microscopy. Cyanidin was rapidly taken up by HaCaT cells at relatively low concentrations. Following incubation, cellular cyanidin levels decreased time-dependently most likely due to degradation into protocatechuic acid and phloroglucinol aldehyde. Confocal laser scanning microscopy data demonstrated that cyanidin was mainly present in the cytoplasm. Cellular uptake of cyanidin was accompanied by an inhibition of multidrug resistance protein 1 (involved in cellular efflux of flavonoids) mRNA-levels indicating its gene-regulatory activity. Naturstoff reagent A seems to be a promising fluorescent dye to visualize cyanidin in keratinocytes.

Ochratoxin A impairs Nrf2-dependent gene expression in porcine kidney tubulus cells.

The mycotoxin, ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is frequently present in feedstuffs. Ochratoxin A exhibits a wide range of toxic activities including nephrotoxicity. However, the underlying molecular mechanisms of OTA-induced cellular nephrotoxicity have yet not been fully elucidated. Nrf2 is a basic leucine zipper transcriptional activator essential for the coordinated transcriptional induction of antioxidant and xenobiotic metabolizing enzymes in the kidney. Therefore, in the present study, the effects of OTA on the nuclear translocation and transactivation of the transcription factor Nrf2 as well as mRNA levels of Nrf2 target genes including glutathione-S-transferase and gamma-glutamylcysteinyl synthetase have been studied in cultured porcine kidney tubulus cells (LLC-PK1). Nrf2 was induced by sulforaphane, a well-known activator of this transcription factor. Ochratoxin A significantly decreased gamma-glutamylcysteinyl synthetase and glutathione-S-transferase mRNA levels in LLC-PK1 cells. Decreased mRNA levels of gamma-glutamylcysteinyl synthetase and glutathione-S-transferase were accompanied by a lowered nuclear translocation and transactivation of Nrf2. Furthermore, OTA also lowered Nrf2 mRNA levels. Current data indicate that OTA nephrotoxicity may be, at least partly, mediated by an Nrf2-dependent signal transduction pathway.

Dexamethasone impairs hypoxia-inducible factor-1 function.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of alpha- and beta-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1alpha levels in the cytosol of HepG2 cells, while nuclear HIF-1alpha levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

Effects of diazepam and flumazenil on minimum alveolar concentrations for dogs anesthetized with isoflurane or a combination of isoflurane and fentanyl.

OBJECTIVE: To determine the effect of a constant-rate infusion of fentanyl on minimum alveolar concentration (MAC) of isoflurane and to determine the interaction between fentanyl and a benzodiazepine agonist (diazepam) and antagonist (flumazenil) in isoflurane-anesthetized dogs. ANIMALS: 8 mixed-breed adult dogs. PROCEDURE: Dogs were anesthetized with isoflurane 3 times during a 6-week period. After a 30-minute equilibration period, each MAC determination was performed in triplicate, using standard techniques. Fentanyl was administered as a bolus (10 microg/kg of body weight, IV) that was followed by a constant infusion (0.3 microg/kg per min, IV) throughout the remainder of the experiment. After determining isoflurane-fentanyl MAC in triplicate, each dog received saline (0.9% NaCl) solution, diazepam, or flumazenil. After 30 minutes, MAC was determined again. RESULTS: Fentanyl significantly decreased isoflurane MAC (corrected to a barometric pressure of 760 mm Hg) from 1.80+/-0.21 to 0.85+/-0.14%, a reduction of 53%. Isoflurane-fentanyl-diazepam MAC (0.48+/-0.29%) was significantly less than isoflurane-fentanyl-saline MAC (0.79+/-0.21%). Percentage reduction in isoflurane MAC was significantly greater for fentanyl-diazepam (74%), compared with fentanyl-saline (54%) or fentanyl-flumazenil (61%). Mean fentanyl concentrations for the entire experiment were increased over time and were higher in the diazepam group than the saline or flumazenil groups. CONCLUSIONS AND CLINICAL RELEVANCE: Fentanyl markedly decreased isoflurane MAC in dogs. Diazepam, but not flumazenil, further decreased isoflurane-fentanyl MAC. Our results indicate that diazepam enhances, whereas flumazenil does not affect, opioid-induced CNS depression and, possibly, analgesia in dogs.

Changes in anesthetic sensitivity and glutamate receptors in the aging canine brain.

This study investigated whether the aging process in dogs is associated with an increased sensitivity to inhalation anesthesia and whether age-related changes in glutamate receptors are related to the increased sensitivity. The mean minimum alveolar concentration (MAC) of isoflurane was 1.82 +/- .08% for 2-3 year olds and 1.45 +/- .06% for 11 years olds, indicating that there was an increased potency of isoflurane in the older dogs as compared to the young. These older animals also showed a significant decrease in binding of [3H]glutamate and [3H]dizocilpine ([3H]MK801) to N-methyl-D-aspartate (NMDA) receptors in multiple cortical and hippocampal regions. The density of binding to NMDA receptors in the cortex, using a single concentration of ligand, correlated significantly with individual MAC values. These results demonstrate that dogs experience an increase in anesthetic potency with increased age, similar to humans, and that age-related changes in the NMDA receptor may represent one mechanism underlying this increased sensitivity to anesthesia.

Neurodevelopmental outcome in ECMO vs near-miss ECMO patients at 5 years of age.

The objective of this study was to compare the outcome of children at 5 years of age who were treated with extracorporeal membrane oxygenation (ECMO) and those who were critically ill but did not meet ECMO criteria, identified as near-miss ECMO. In one of the longest studies of its kind, we compared the neurodevelopmental outcome of 76 5-year-old ECMO-treated children with 20 5-year-old near-miss ECMO patients with similar primary diagnoses. The two groups were compared for demographic data, level of ventilatory support, and degree of hyperventilation. The comprehensive assessment protocol included an assessment of intelligence (IQ), attainment of preacademic and early academic skills, and parents' report of adaptive behavior. Both groups had similar demographic data and primary diagnosis. The near-miss ECMO patients required increased ventilatory support but not significantly more than the ECMO patients. The cognitive outcome was similar in both groups with mean estimated Full-Scale IQ in the normal range for near-miss and ECMO groups (89 and 97, respectively). Rates of severe mental handicap (FSIQ < 70) (near-miss = 11%, ECMO = 12%) and risk for school failure (near-miss = 38%, ECMO = 37%) were also similar. More parents of near-miss ECMO patients reported immature adaptive skills than did parents of ECMO patients, although the numbers were small in each group. Rates of parent-reported child behavior problems were similar in both groups. ECMO and near-miss ECMO patients have similar cognitive and adaptive outcomes at 5 years of age. A significant number in each group are at risk of school failure and should be closely followed up.